Sulfated glycosaminoglycans such as heparin and the related heparan sulfate (HSGAGs) are complex, linear carbohydrates possessing considerable chemical heterogeneity (Esko, J. D., and Lindahl, U. (2001) J Clin Invest 108(2), 169–73, Shriver, Z., Liu, D., and Sasisekharan, R. (2002) Trends Cardiovasc Med 12(2), 71–72). Their structural diversity is largely a consequence of the variable number and position of sulfates present within a single HSGAG chain. Because of their highly anionic character, these polysaccharides historically have been relegated to an exclusively structural role, namely as a sort of hydration gel and scaffold comprising the extracellular matrix (ECM). Contrary to this limited perception, however, HSGAGs actually play an important and dynamic function in many critical biological processes ranging from development (Perrimon, N., and Bernfield, M. (2000) Nature 404(6779), 725–8) and tissue repair (Simeon, A., Wegrowski, Y., Bontemps, Y., and Maquart, F. X. (2000) J Invest Dermatol 115(6), 962–8) to apoptosis (Ishikawa, Y., and Kitamura, M. (1999) Kidney Int 56(3), 954–63, Kapila, Y. L., Wang, S., Dazin, P., Tafolla, E., and Mass, M. J. (2002) J Biol Chem 277(10), 8482–91). These polysaccharides are also central players in several pathological conditions such as cancer (Selva, E. M., and Perrimon, N. (2001) Adv Cancer Res 83, 67–80, Sasisekharan, R., Shriver, Z., Venkataraman, G., and Narayanasami, U. (2002) Nat Rev Cancer 2(7), 521–8), angiogenesis (Folkman, J., and Shing, Y. (1992) Adv Exp Med Biol 313, 355–64, Vlodavsky, I., Elkin, M., Pappo, O., Aingorn, H., Atzmon, R., Ishai-Michaeli, R., Aviv, A., Pecker, I., and Friedmann, Y. (2000) Isr Med Assoc J 2 Suppl, 37–45), certain neurodegenerative diseases such as Alzheimers (Cohlberg, J. A., Li, J., Uversky, V. N., and Fink, A. L. (2002) Biochemistry 41(5), 1502–11), athleroscelerosis (Sehayek, E., Olivecrona, T., Bengtsson-Olivecrona, G., Vlodavsky, I., Levkovitz, H., Avner, R., and Eisenberg, S. (1995) Atherosclerosis 114(1), 1–8), and microbial infectivity (Liu, J., and Thorp, S. C. (2002) Med Res Rev 22(1), 1–25). HSGAGs do so as part of proteoglycans found at the cell surface and within the ECM where they mediate signaling pathways and cell-cell communication by modulating the bioavailability and temporal-spatial distribution of growth factors, cytokines, and morphogens (Tumova, S., Woods, A., and Couchman, J. R. (2000) Int J Biochem Cell Biol 32(3), 269–88) in addition to various receptors and extracellular adhesion molecules (Lyon, M., and Gallagher, J. T. (1998) Matrix Biol 17(7), 485–93). HSGAG structure and function are inextricably related.
A study of the HSGAG structure-function paradigm (Gallagher, J. T. (1997) Biochem Soc Trans 25(4), 1206–9) requires the ability to determine both the overall composition of biologically relevant HSGAGs as well as ultimately ascertaining their actual linear sequence (fine structure). Therefore the availability of several chemical and enzymatic reagents which are able to cleave HSGAGs in a structure-specific fashion have proven to be valuable. One example of an important class of GAG degrading enzymes is the heparin lyases (heparinases) originally isolated from the gram negative soil bacterium F. heparinum (Ernst, S., Langer, R., Cooney, C. L., and Sasisekharan, R. (1995) Crit Rev Biochem Mol Biol 30(5), 387–444). Each of the three heparinases encoded by this microorganism cleave both heparin and heparan sulfate with a substrate specificity that is generally based on the differential sulfation pattern which exists within each GAG chain (Ernst, S., Langer, R., Cooney, C. L., and Sasisekharan, R. (1995) Crit Rev Biochem Mol Biol 30(5), 387–444, Rhomberg, A. J., Ernst, S., Sasisekharan, R., and Biemann, K. (1998) Proc Natl Acad Sci USA 95(8), 4176–81). In fact, F. heparinum uses several additional enzymes in an apparently sequential manner to first depolymerize and then subsequently desulfate heparin/heparan sulfate. In addition to heparinase I (Sasisekharan, R., Bulmer, M., Moremen, K. W., Cooney, C. L., and Langer, R. (1993) Proc Natl Acad Sci USA 90(8), 3660–4), we have recently cloned one of these enzymes, the Δ 4,5 unsaturated glycuronidase (Myette, J. R., Shriver, Z., Kiziltepe, T., McLean, M. W., Venkataraman, G., and Sasisekharan, R. (2002) Biochemistry 41(23), 7424–7434). This enzyme has been recombinantly expressed in E. coli as a highly active enzyme. Because of its rather unique substrate specificity (Wamick, C. T., and Linker, A. (1972) Biochemistry 11(4), 568–72), this enzyme has already proven to be a useful addition to our PEN-MALDI based carbohydrate sequencing methodology (Venkataraman, G., Shriver, Z., Raman, R., and Sasisekharan, R. (1999) Science 286(5439), 537–42).